The structure of synapses is dynamic-once formed, neural circuits can evolve by the addition and elimination of synaptic connections. This plasticity underlies the refinement of developing circuits and defects in these synaptic growth mechanisms are a likely etiology of neurodevelopmental disorders such as autism, mental retardation, and epilepsy. In the mature nervous system, such morphological plasticity is likely important for normal processes such as memory formation and pathophysiological events such as the synaptic rearrangements related to drug addiction. To define the molecular mechanisms that regulate synaptic growth, we have undertaken a genetic analysis in Drosophila. We have identified highwire as the most potent inhibitor of synaptic growth yet identified in Drosophila. In its absence, there is a large increase in the number of synaptic boutons and branches, while each bouton is smaller and synaptic transmission is impaired. We hypothesize that highwire is a central component of an evolutionarily conserved signaling pathway controlling synaptic growth. In this proposal we will define the molecular mechanisms by which highwire inhibits synaptic growth while facilitating neurotransmitter release. Toward these ends, we have generated highwire transgenes that fully rescue the morphological and functional phenotypes of the highwire mutant. These provide the basis for conducting a structure/function analysis of this large, multidomain protein (Aim 1). We have identified biochemical and genetic interactors with highwire and we will characterize their function for the regulation of synaptic growth (Aim 2). Finally, we have demonstrated that the mixed lineage kinase wallenda is necessary for synaptic overgrowth in a highwire mutant. We will investigate the function of this kinase-signaling pathway for the control of synaptic growth (Aim 3). This research is relevant to public health because it will improve our understanding of how nerve cells connect with each other in the brain. If these connections do not form properly in a child it may lead to neurological diseases such as autism, mental retardation, and epilepsy. In the adult, inappropriate changes in these connections may contribute to diseases such as chronic pain and drug addiction. An understanding of the molecules that control the growth of nerve cell connections could aid in the future development of new therapies for devastating neurological diseases.